Cardiac glycosides are steroidal compounds of plant origin that enjoy widespread use in medicine and the basic sciences. They bind with high affinity and specificity to a highly conserved site on the sodium-potassium pump. Overwhelming structural and functional evidence shows the presence of an isomer of ouabain in the circulation, and tissues of mammals including man and cattle. Much evidence suggests that the adrenal cortex may be the primary source of the circulating isomer (i.e., an "adrenal derived endogenous ouabain, AEO). However, the biosynthesis of AEO by mammals has not been described. The successful outcome of this investigation may constitute a major advance in steroid biochemistry and lend further credence to the evidence that AEO and related steroids are of physiological and medical significance. The proposal describes experiments using established procedures that investigate the synthesis of AEO by bovine adrenocortical tissue and cells in culture. The aims are: 1) to determine whether common steroidogenic precursors support synthesis and adrenal secretion of AEO and aldosterone; 2) to use radiolabeled precursors to determine whether they can be transformed by an ordered sequential process to AEO, and the point at which the pathway diverges from that for aldosterone; 3) to determine the functional activity of the secreted materials by analysis of the binding and inhibitory mechanism of action on the sodium pump, and 4) to investigate the structure of the secreted AEO and stable biologically active precursors using mass spectral and nuclear magnetic resonance techniques. Detailed knowledge of the structure of the adrenal materials can provide new insights into the design of improved cardiotonics as well as novel agents that interfere with receptor binding and adrenal biosynthesis.